Lettuce variety red crispita

ABSTRACT

The present invention provides novel lettuce cultivar Red Crispita and plant parts, seed, and tissue culture therefrom. The invention also provides methods for producing a lettuce plant by crossing the lettuce plants of the invention with themselves or another lettuce plant. The invention also provides lettuce plants produced from such a crossing as well as plant parts, seed, and tissue culture therefrom.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(f) fromNetherlands Plant Variety Rights application No. SLA3835, filed May 8,2017.

FIELD OF THE INVENTION

This invention is in the field of lettuce plants.

BACKGROUND OF THE INVENTION

The present invention relates to a red leaf lettuce (Lactuca sativa L.)variety designated Red Crispita.

Practically speaking, all cultivated forms of lettuce belong to thehighly polymorphic species Lactuca sativa that is grown for its ediblehead and leaves. Lactuca sativa is in the Cichoreae tribe of theAsteraceae (Compositae) family. Lettuce is related to chicory,sunflower, aster, dandelion, artichoke, and chrysanthemum. Sativa is oneof about 300 species in the genus Lactuca. There are seven differentmorphological types of lettuce. The crisphead group includes the icebergand batavian types. Iceberg lettuce has a large, firm head with a crisptexture and a white or creamy yellow interior. The batavian lettucepredates the iceberg type and has a smaller and less firm head. Thebutterhead group has a small, soft head with an almost oily texture. Theromaine, also known as cos lettuce, has elongated upright leaves forminga loose, loaf-shaped head and the outer leaves are usually dark green.Leaf lettuce comes in many varieties, none of which form a head, andinclude the green oak leaf variety. Latin lettuce looks like a crossbetween romaine and butterhead. Stem lettuce has long, narrow leaves andthick, edible stems. Oilseed lettuce is a type grown for its large seedsthat are pressed to obtain oil. Latin lettuce, stem lettuce, and oilseedlettuce are seldom seen in the United States.

Presently, there are over one thousand known lettuce cultivars. As acrop, lettuce is grown commercially wherever environmental conditionspermit the production of an economically viable yield.

Lettuce, in general, and leaf lettuce in particular, is an important andvaluable vegetable crop. Thus, there is an ongoing need for improvedlettuce varieties.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel lettuce cultivardesignated Red Crispita, also known as LS16037, which is a baby leaflettuce and has characteristics including red mature leaf color,exceptional leaf thickness and crispiness of the variety as comparedwith standard red leaf varieties, and resistances to Bremia lactucaeraces 1-18 and 20-32. The invention also encompasses the seeds oflettuce cultivar Red Crispita, the plants of lettuce cultivar RedCrispita, plant parts of the lettuce cultivar Red Crispita (includingleaves, seed, gametes), methods of producing seed from lettuce cultivarRed Crispita, and method for producing a lettuce plant by crossing thelettuce cultivar Red Crispita with itself or another lettuce plant,methods for producing a lettuce plant containing in its genetic materialone or more transgenes, and the transgenic lettuce plants produced bythat method. The invention also relates to methods for producing otherlettuce plants derived from lettuce cultivar Red Crispita and to lettuceplants, parts thereof and seed derived by the use of those methods. Thepresent invention further relates to hybrid lettuce seeds and plants(and parts thereof including leaves) produced by crossing lettucecultivar Red Crispita with another lettuce plant.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of lettuce cultivar Red Crispita. In embodiments,the tissue culture is capable of regenerating plants having all oressentially all of the physiological and morphological characteristicsof the foregoing lettuce plant and/or of regenerating plants having thesame or substantially the same genotype as the foregoing lettuce plant.In exemplary embodiments, the regenerable cells in such tissue culturesare meristematic cells, cotyledons, hypocotyl, leaves, pollen, embryos,roots, root tips, anthers, pistils, ovules, shoots, stems, petiole,pith, flowers, capsules and/or seeds as well as callus and/orprotoplasts derived from any of the foregoing. Still further, thepresent invention provides lettuce plants regenerated from the tissuecultures of the invention.

As a further aspect, the invention provides a method of producinglettuce seed, the method comprising crossing a plant of lettuce cultivarRed Crispita with itself or a second lettuce plant. Optionally, themethod further comprises collecting the seed.

Another aspect of the invention provides methods for producing hybridsand other lettuce plants derived from lettuce cultivar Red Crispita.Lettuce plants derived by the use of those methods are also part of theinvention as well as plant parts, seed, gametes and tissue culture fromsuch hybrid or derived lettuce plants.

In representative embodiments, a lettuce plant derived from lettucecultivar Red Crispita comprises cells comprising at least one set ofchromosomes derived from lettuce cultivar Red Crispita. In embodiments,a lettuce plant or population of lettuce plants derived from lettucecultivar Red Crispita comprises, on average, at least 6.25%, 12.5%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% of its alleles (i.e., theoretical allelic content; TAC)from lettuce cultivar Red Crispita, e.g., at least about 6.25%, 12.5%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% of the genetic complement of lettuce cultivar RedCrispita, and optionally is the result of a breeding process comprisingone or two breeding crosses and one or more of selfing, sibbing,backcrossing and/or double haploid techniques in any combination and anyorder. In embodiments, the breeding process does not include a breedingcross, and comprises selfing, sibbing, backcrossing and or doublehaploid technology. In embodiments, the lettuce plant derived fromlettuce cultivar Red Crispita is one, two, three, four, five or morebreeding crosses removed from lettuce cultivar Red Crispita.

In embodiments, a hybrid or derived plant from lettuce cultivar RedCrispita comprises a desired added trait(s). In representativeembodiments, a lettuce plant derived from lettuce cultivar Red Crispitacomprises all of the morphological and physiological characteristics oflettuce cultivar Red Crispita (e.g., as described in Table 1 and/orTables 2 to 5, for example, red mature leaf color, thick and crispleaves, and highly resistant to Bremia lactucae races 1-18 and 20-32).In embodiments, the lettuce plant derived from lettuce cultivar RedCrispita comprises essentially all of the morphological andphysiological characteristics of lettuce cultivar Red Crispita (e.g., asdescribed in Table 1 and/or Tables 2 to 5, for example, red mature leafcolor, thick and crisp leaves, and highly resistant to Bremia lactucaeraces 1-18 and 20-32), with the addition of a desired added trait(s).

The invention also relates to methods for producing a lettuce plantcomprising in its genetic material one or more transgenes and to thetransgenic lettuce plant produced by those methods (and progeny lettuceplants comprising the transgene). Also provided are plant parts, seedand tissue culture from such transgenic lettuce plants, optionallywherein one or more cells in the plant part, seed, or tissue culturecomprises the transgene. The transgene can be introduced via planttransformation and/or breeding techniques.

In another aspect, the present invention provides for single geneconverted plants of lettuce cultivar Red Crispita. Plant parts, seed,and tissue culture from such single gene converted plants are alsocontemplated by the present invention. The single transferred gene maybe a dominant or recessive allele. In representative embodiments, thesingle transferred gene confers such traits as male sterility, herbicideresistance, pest resistance (e.g., insect and/or nematode resistance),modified fatty acid metabolism, modified carbohydrate metabolism,disease resistance (e.g., for bacterial, fungal and/or viral disease),male fertility, enhanced nutritional quality, improved appearance (e.g.,color), improved salt tolerance, industrial usage, or any combinationthereof. The single gene may be a naturally occurring lettuce gene or atransgene introduced into lettuce through genetic engineeringtechniques.

The invention further provides methods for developing lettuce plants ina lettuce plant breeding program using plant breeding techniquesincluding, for example, recurrent selection, backcrossing, pedigreebreeding, double haploid techniques, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selectionand/or transformation. Seeds, lettuce plants, and parts thereof,produced by such breeding methods are also part of the invention.

The invention also provides methods of multiplication or propagation oflettuce plants of the invention, which can be accomplished using anymethod known in the art, for example, via vegetative propagation and/orseed.

The invention further provides a method of producing food or feedcomprising (a) obtaining a lettuce plant of the invention, optionallywherein the plant has been cultivated to maturity, and (b) collecting atleast one lettuce plant or part thereof (e.g., leaves) from the plant.

Additional aspects of the invention include harvested products andprocessed products from the lettuce plants of the invention. A harvestedproduct can be a whole plant or any plant part, as described herein.Thus, in some embodiments, a non-limiting example of a harvested productincludes a seed, a leaf and/or a stem.

In representative embodiments, a processed product includes, but is notlimited to: cut, sliced, ground, pureed, dried, canned, jarred, washed,packaged, frozen and/or heated leaves and/or seeds of the lettuce plantsof the invention, or any other part thereof. In embodiments, a processedproduct includes a sugar or other carbohydrate, fiber, protein and/oraromatic compound that is extracted, purified or isolated from a lettuceplant of the invention. In embodiments, the processed product includeswashed and packaged leaves (or parts thereof) of the invention.

The seed of the invention can optionally be provided as an essentiallyhomogenous population of seed of a single plant or cultivar. Essentiallyhomogenous populations of seed are generally free from substantialnumbers of other seed, e.g., at least about 90%, 95%, 96%, 97%, 98% or99% pure.

In representative embodiments, the invention provides a seed of lettucecultivar Red Crispita.

As a further aspect, the invention provides a plant of lettuce cultivarRed Crispita.

As an additional aspect, the invention provides a lettuce plant, or apart thereof, having all or essentially all of the physiological andmorphological characteristics of a plant of lettuce cultivar RedCrispita.

As another aspect, the invention provides leaves and/or seed of thelettuce plants of the invention and a processed product from the leavesand/or seed of the inventive lettuce plants.

As still another aspect, the invention provides a method of producinglettuce seed, the method comprising crossing a lettuce plant of theinvention with itself or a second lettuce plant. The invention alsoprovides seed produced by this method and plants produced by growing theseed.

As yet a further aspect, the invention provides a method for producing aseed of a lettuce plant derived from lettuce cultivar Red Crispita, themethod comprising: (a) crossing a lettuce plant of lettuce cultivar RedCrispita with a second lettuce plant; and (b) allowing seed of a lettuceplant derived from lettuce cultivar Red Crispita to form. Inembodiments, the method further comprises: (c) growing a plant from theseed derived from lettuce cultivar Red Crispita of step (b); (d) selfingthe plant grown from the lettuce seed derived from lettuce cultivar RedCrispita or crossing it to a second lettuce plant to form additionallettuce seed derived from lettuce cultivar Red Crispita, and (e)repeating steps (c) and (d) 0 or more times to generate further derivedlettuce seed. Optionally, the method comprises: (e) repeating steps (c)and (d) one or more times (e.g., one to three, one to five, one to six,one to seven, one to ten, three to five, three to six, three to seven,three to eight or three to ten times) to generate further derivedlettuce plants. As another option, the method can comprise collectingthe seed. The invention also provides seed produced by these methods andplants produced by growing the seed.

As another aspect, the invention provides a method of producing lettuceleaves, the method comprising: (a) obtaining a plant of lettuce cultivarRed Crispita, optionally wherein the plant has been cultivated tomaturity; and (b) collecting leaves from the plant. The invention alsoprovides the leaves produced by this method.

Still further, as another aspect, the invention provides a method ofvegetatively propagating a plant of lettuce cultivar Red Crispita. In anon-limiting example, the method comprises: (a) collecting tissuecapable of being propagated from a plant of lettuce cultivar RedCrispita; (b) cultivating the tissue to obtain proliferated shoots; and(c) rooting the proliferated shoots to obtain rooted plantlets.Optionally, the invention further comprises growing plants from therooted plantlets. The invention also encompasses the plantlets andplants produced by these methods.

As an additional aspect, the invention provides a method of introducinga desired added trait into lettuce cultivar Red Crispita, the methodcomprising: (a) crossing a first plant of lettuce cultivar Red Crispitawith a second lettuce plant that comprises a desired trait to produce F₁progeny; (b) selecting an F₁ progeny that comprises the desired trait;(c) crossing the selected F₁ progeny with lettuce cultivar Red Crispitato produce backcross progeny; and (d) selecting backcross progenycomprising the desired trait to produce a plant derived from lettucecultivar Red Crispita comprising a desired trait. In embodiments, theselected progeny comprises all or essentially all the morphological andphysiological characteristics of the first plant of lettuce cultivar RedCrispita. Optionally, the method further comprises: (e) repeating steps(c) and (d) one or more times in succession (e.g., one to three, one tofive, one to six, one to seven, one to ten, three to five, three to six,three to seven, three to eight or three to ten times) to produce a plantderived from lettuce cultivar Red Crispita comprising the desired trait.

In representative embodiments, the invention also provides a method ofproducing a plant of lettuce cultivar Red Crispita comprising a desiredadded trait, the method comprising introducing a transgene conferringthe desired trait into a plant of lettuce cultivar Red Crispita. Thetransgene can be introduced by transformation methods (e.g., geneticengineering) or breeding techniques. In embodiments, the plantcomprising the transgene comprises all or essentially all of themorphological and physiological characteristics of lettuce cultivar RedCrispita.

The invention also provides lettuce plants produced by the methods ofthe invention, wherein the lettuce plant has the desired added trait aswell as seed from such lettuce plants.

According to the foregoing methods, the desired added trait can be anysuitable trait known in the art including, for example, male sterility,male fertility, herbicide resistance, insect or pest (e.g., insectand/or nematode) resistance, modified fatty acid metabolism, modifiedcarbohydrate metabolism, disease resistance (e.g., for bacterial, fungaland/or viral disease), enhanced nutritional quality, increasedsweetness, increased flavor, improved ripening control, improved salttolerance, industrial usage, or any combination thereof.

In representative embodiments, a transgene conferring herbicideresistance confers resistance to glyphosate, sulfonylurea,imidazolinone, dicamba, glufosinate, phenoxy proprionic acid,L-phosphinothricin, cyclohexone, cyclohexanedione, triazine,benzonitrile, or any combination thereof.

In representative embodiments, a transgene conferring pest resistance(e.g., insect and/or nematode resistance) encodes a Bacillusthuringiensis endotoxin.

In representative embodiments, transgenic plants, transformed plants,hybrid plants and lettuce plants derived from lettuce cultivar RedCrispita have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of themorphological and physiological characteristics of lettuce cultivar RedCrispita (e.g., as described in Table 1 and/or Tables 2 to 5, forexample, red mature leaf color, thick and crisp leaves, and/or highlyresistant to Bremia lactucae races 1-18 and 20-32, or even all of themorphological and physiological characteristics of lettuce cultivar RedCrispita, so that said plants are not significantly different for saidtraits than lettuce cultivar Red Crispita, as determined at the 5%significance level when grown in the same environmental conditions;optionally, with the presence of one or more desired additional traits(e.g., male sterility, disease resistance, pest or insect resistance,herbicide resistance, and the like).

In embodiments, the plants of the invention have at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more of the morphological and physiologicalcharacteristics of lettuce cultivar Red Crispita (e.g., as described inTable 1 and/or Tables 2 to 5). For example, the plants of the inventioncan have one or both of the following characteristics: red mature leafcolor, thick and crisp leaves, and/or highly resistant to Bremialactucae races 1-18 and 20-32.

The invention also encompasses plant parts, plant material, pollen,ovules, leaves, fruit and seed from the lettuce plants of the invention.Also provided is a tissue culture of regenerable cells from the lettuceplants of the invention, where optionally, the regenerable cells are:(a) embryos, meristem, leaves, pollen, cotyledons, hypocotyls, roots,root tips, anthers, flowers, pistils, ovules, seed, shoots, stems,stalks, petioles, pith and/or capsules; or (b) callus or protoplastsderived from the cells of (a). Further provided are lettuce plantsregenerated from a tissue culture of the invention.

In still yet another aspect, the invention provides a method ofdetermining a genetic characteristic of lettuce cultivar Red Crispita ora progeny thereof, e.g., a method of determining a genotype of lettucecultivar Red Crispita or a progeny thereof using molecular genetictechniques. In embodiments, the method comprises detecting in the genomeof a Red Crispita plant, or a progeny plant thereof, at least a firstpolymorphism, e.g., comprises nucleic acid amplification and/or nucleicacid sequencing. To illustrate, in embodiments, the method comprisesobtaining a sample of nucleic acids from the plant and detecting atleast a first polymorphism in the nucleic acid sample (e.g., using oneor more molecular markers). Optionally, the method may comprisedetecting a plurality of polymorphisms (e.g., two or more, three ormore, four or more, five or more, six or more, eight or more or ten ormore polymorphisms, etc.) in the genome of the plant. In representativeembodiments, the method further comprises storing the results of thestep of detecting the polymorphism(s) on a computer readable medium. Theinvention further provides a computer readable medium produced by such amethod.

In addition to the exemplary aspects and embodiments described above,the invention is described in more detail in the description of theinvention set forth below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the development of a novellettuce cultivar having desirable characteristics including red matureleaf color, exceptionally thick and crispy leaves, and high resistanceto Bremia lactucae races 1-18 and 20-32.

It should be appreciated that the invention can be embodied in differentforms and should not be construed as limited to the embodiments setforth herein. Rather, these embodiments are provided so that thisdisclosure will be thorough and complete, and will fully convey thescope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

Unless the context indicates otherwise, it is specifically intended thatthe various features and embodiments of the invention described hereincan be used in any combination.

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted. To illustrate, if thespecification states that a composition comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed singularly or in any combination.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Definitions

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

The term “about,” as used herein when referring to a measurable valuesuch as a dosage or time period and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

The term “comprise,” “comprises” and “comprising” as used herein,specify the presence of the stated features, integers, steps,operations, elements, and/or components, but do not preclude thepresence or addition of one or more other features, integers, steps,operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase “consisting essentially of”means that the scope of a claim is to be interpreted to encompass thespecified materials or steps recited in the claim “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463(CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus,the term “consisting essentially of” when used in a claim or thedescription of this invention is not intended to be interpreted to beequivalent to “comprising.”

“Allele”. An allele is any of one or more alternative forms of a gene,all of which relate to a trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

“Backcrossing”. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

“Big Vein virus”. Big vein is a disease of lettuce caused by LettuceMirafiori Big Vein Virus which is transmitted by the fungus Olpidiumvirulentus, with vein clearing and leaf shrinkage resulting in plants ofpoor quality and reduced marketable value.

“Bolting”. The premature development of a flowering stalk, andsubsequent seed, before a plant produces a food crop. Bolting istypically caused by late planting when temperatures are low enough tocause vernalization of the plants.

“Bremia lactucae”. An Oomycete that causes downy mildew in lettuce incooler growing regions.

“Core length”. Length of the internal lettuce stem measured from thebase of the cut and trimmed head to the tip of the stem.

“Corky root”. A disease caused by the bacterium Sphingomonassuberifaciens, which causes the entire taproot to become brown, severelycracked, and non-functional.

“Cotyledon”. One of the first leaves of the embryo of a seed plant;typically one or more in monocotyledons, two in dicotyledons, and two ormore in gymnosperms.

“Double haploid line”. A stable inbred line achieved by doubling thechromosomes of a haploid line, e.g., from anther culture. For example,some pollen grains (haploid) cultivated under specific conditionsdevelop plantlets containing 1 n chromosomes. The chromosomes in theseplantlets are then induced to “double” (e.g., using chemical means)resulting in cells containing 2n chromosomes. The progeny of theseplantlets are termed “double haploid” and are essentially notsegregating any more (e.g., are stable). The term “double haploid” isused interchangeably herein with “dihaploid.”

“Essentially all of the physiological and morphologicalcharacteristics”. In embodiments, a plant having “essentially all of thephysiological and morphological characteristics” (and similar phrases)means a plant having the physiological and morphological characteristicsof the recurrent parent, except for the characteristics derived from theconverted gene(s). In embodiments, a plant having “essentially all ofthe physiological and morphological characteristics” (and similarphrases) means a plant having all of the characteristics of thereference plant with the exception of five or fewer traits, 4 or fewertraits, 3 or fewer traits, 2 or fewer traits, or one trait. According tothe foregoing embodiments, a plant having “essentially all of thephysiological and morphological characteristics” (and similar phrases)optionally has a red mature leaf color, thick and crisp leaves, and/oris highly resistant to Bremia lactucae races 1-18 and 20-32.

“First water date”. The date the seed first receives adequate moistureto germinate. This can and often does equal the planting date.

“Gene”. As used herein, “gene” refers to a segment of nucleic acidcomprising an open reading frame. A gene can be introduced into a genomeof a species, whether from a different species or from the same species,using transformation or various breeding methods.

“Head diameter”. Diameter of the cut and trimmed head, slicedvertically, and measured at the widest point perpendicular to the stem.

“Head height”. Height of the cut and trimmed head, sliced vertically,and measured from the base of the cut stem to the cap leaf.

“Head weight”. Weight of saleable lettuce head, cut and trimmed tomarket specifications.

“Inbred line”: As used herein, the phrase “inbred line” refers to agenetically homozygous or nearly homozygous population. An inbred line,for example, can be derived through several cycles of sib crossingand/or selfing and/or via double haploid production. In someembodiments, inbred lines breed true for one or more traits of interest.An “inbred plant” or “inbred progeny” is an individual sampled from aninbred line.

“Lettuce Mosaic virus”. A disease that can cause a stunted, deformed, ormottled pattern in young lettuce and yellow, twisted, and deformedleaves in older lettuce.

“Maturity date”. Maturity refers to the stage when the plants are offull size and/or optimum weight and/or in marketable form to be ofcommercial or economic value.

“Nasonovia ribisnigri”. A lettuce aphid that colonizes the innermostleaves of the lettuce plant, contaminating areas that cannot be treatedeasily with insecticides.

“Plant.” As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as leaves, pollen, embryos,cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers,ovules, seeds, fruit, stems, and the like.

“Plant material”. The terms “plant material” and “material obtainablefrom a plant” are used interchangeably herein and refer to any plantmaterial obtainable from a plant including without limitation, leaves,stems, roots, flowers or flower parts, fruits, pollen, ovules, zygotes,seeds, cuttings, cell or tissue cultures, or any other part or productof the plant.

“Plant part”. As used herein, a “plant part” includes any part, organ,tissue or cell of a plant including without limitation an embryo,meristem, leaf, pollen, cotyledon, hypocotyl, root, root tip, anther,flower, flower bud, pistil, ovule, seed, shoot, stem, stalk, petiole,pith, capsule, a scion, a rootstock and/or a fruit including callus andprotoplasts derived from any of the foregoing.

“Quantitative Trait Loci”. Quantitative Trait Loci (QTL) refers togenetic loci that control to some degree, numerically representabletraits that are usually continuously distributed.

“Ratio of head height/diameter”. Head height divided by the headdiameter is an indication of the head shape; <1 is flattened, 1=round,and >1 is pointed.

“Regeneration”. Regeneration refers to the development of a plant fromtissue culture.

“Resistance”. As used herein the terms “resistance” and “tolerance” (andgrammatical variations thereof) are used interchangeably to describeplants that show reduced or essentially no symptoms to a specific biotic(e.g., a pest, pathogen or disease) or abiotic (e.g., exogenous orenvironmental, including herbicides) factor or stressor. In someembodiments, “resistant” or “tolerant” plants show some symptoms but arestill able to produce marketable product with an acceptable yield, e.g.,the yield may still be reduced and/or the plants may be stunted ascompared with the yield or growth in the absence of the biotic and/orabiotic factor or stressor. Those skilled in the art will appreciatethat the degree of resistance or tolerance may be assessed with respectto a plurality or even an entire field of plants. A lettuce plant may beconsidered “resistant” or “tolerant” if resistance/tolerance is observedover a plurality of plants (e.g., an average), even if particularindividual plants may be susceptible to the biotic or abiotic factor orstressor.

“RHS”. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Horticulture Society Enterprise Ltd., RHS Garden;Wisley, Woking; Surrey GU236QB, UK.

“Single gene converted”. A single gene converted or conversion plantrefers to a plant that is developed by plant breeding techniques (e.g.,backcrossing) or via genetic engineering wherein essentially all of thedesired morphological and physiological characteristics of a line arerecovered in addition to the single gene transferred into the line viathe plant breeding technique or via genetic engineering.

“Substantially equivalent characteristic”. A characteristic that, whencompared, does not show a statistically significant difference (e.g.,p=0.05) from the mean.

“Tip burn”. Means a browning of the edges or tips of lettuce leaves thatis a physiological response to a lack of calcium.

“Tomato Bushy Stunt”. Also called “lettuce necrotic stunt”. A diseasethat causes stunting of growth and leaf mottling.

“Transgene”. A nucleic acid of interest that can be introduced into thegenome of a plant by genetic engineering techniques (e.g.,transformation) or breeding. The transgene can be from the same or adifferent species. If from the same species, the transgene can be anadditional copy of a native coding sequence or can present the nativesequence in a form or context (e.g., different genomic location and/orin operable association with exogenous regulatory elements such as apromoter) than is found in the native state. The transgene can comprisean open reading frame encoding a polypeptide or can encode a functionalnon-translated RNA (e.g., RNAi).

Botanical Description of the Lettuce Cultivar Red Crispita (LS16037).

Characteristics. Red Crispita is a red babyleaf variety. Lettuce varietyRed Crispita resulted from a cross between a commercial lettuce varietyand a proprietary breeding line and subsequent numerous generations ofindividual plant selections chosen for their red color and desirablehorticultural traits. Red Crispita is characterized by a red mature leafcolor as well as thick and crispy leaves as compared with standard redleaf varieties.

Red Crispita has shown uniformity and stability for the traits, withinthe limits of environmental influence. It has been self-pollinated asufficient number of generations with careful attention to uniformity ofplant type. The variety has been increased with continued observationfor uniformity.

TABLE 1 Variety Description Information. Plant Type: Red Babyleaf SeedSeed color: White Seed length: Medium Seedling Seedling, anthocyanincoloration: Present Size of cotyledon: Medium Shape of cotyledon: Narrowelliptic Mature Leaves Hue of green color of outer leaves: Reddish Colorof outer leaves (at harvest maturity): Reddish Intensity of color ofouter leaves: Medium to Dark Anthocyanin coloration: Present Intensityof anthocyanin coloration: Strong to Very strong Glossiness of upperside: Medium Leaf blistering: Very weak to Weak Size of Blisters: Verysmall to Small Leaf thickness: Medium to Thick Leaf shape: Obovate Leafblade, incision of margin on apical part: Absent Leaf blade, degree ofundulation of margin: Absent or Very weak Plant Head shape: Head absentPlant type: Cutting Plant size: Very small to Small Intensity offasciation (flowering plant): Absent Time of beginning of bolting (underlong day conditions): Late Bolting behavior comparable with: SadawiDisease and Stress Reactions Virus Lettuce Mosaic virus strain Ls-1:Susceptible Fungal/Bacterial Bremia lactucae, races 1-18, 20-32: Highlyresistant Fusarium oxysporum f. sp. lactucae, races 1 and 2: Not testedInsects Root Aphids: Not tested Nasonovia ribisnigri biotype Nr: 0:Susceptible

Red Crispita in comparison with:

Sate of State of Comparison Expression of Expression of VarietyCharacteristic Comparison Variety Candidate Variety Crispita Leaf:anthocyanin Absent Present coloration Redflash Seed color Black White

TABLE 2 The length of the cotyledon leaf measured in 20-day-oldseedlings (mm) Red Crispita Haflex Telex (LS16037) 1.4 1.1 1.7 1.8 1.31.8 1.2 1.5 1.9 1.5 1.7 1.3 1.3 1.5 1.5 1.4 2.0 1.5 1.5 1.8 1.5 1.3 1.01.7 1.5 1.5 1.9 1.3 1.4 1.8 1.5 1.3 1.7 1.3 1.5 1.8 1.0 1.7 1.7 1.5 1.51.7 1.4 1.3 2.0 1.5 1.3 1.6 1.2 1.5 1.7 1.2 1.8 1.3 1.5 1.7 1.9 1.2 1.51.9 ANOVA: Single factor Summary Groups Count Sum Average VarianceHalfex 20 27.5 1.38 0.032 Telex 20 29.9 1.50 0.054 Red Crispita 20 33.91.70 0.041 ANOVA Source of F critical Variation SS df MS F p value(2,57) Between Varieties 1.045 2 0.523 12.23 <0.0001 3.158 WithinVarieties 2.437 57 0.043 Total 3.482 59 Anova shows there is asignificant difference in the length of the cotyledon leaf measured in20-day-old seedlings.

TABLE 3 The width of the cotyledon leaf measured in 20-day-old seedlings(cm) Red Crispita Haflex Telex (LS16037) 0.9 0.7 0.8 0.7 0.7 0.7 0.7 0.90.8 0.9 0.8 0.7 0.7 0.8 0.7 0.9 0.7 0.7 1 0.9 0.6 0.9 0.8 0.8 0.4 0.80.7 1 0.8 0.7 1 0.8 0.6 0.8 0.7 0.8 0.9 0.8 0.7 0.7 0.8 0.7 0.8 0.9 0.90.9 0.8 0.7 1 0.8 0.6 0.8 0.7 0.7 0.8 0.8 0.7 0.8 0.8 0.6 ANOVA: Singlefactor Summary Groups Count Sum Average Variance Halfex 20 16.6 0.830.0212 Telex 20 15.8 0.79 0.0041 Red Crispita 20 14.2 0.71 0.0062 ANOVAF critical Source DF SS MS F Prob > F (2,57) Between 2 0.149 0.07477.117 0.0017 3.158 Varieties Within 57 0.598 0.0105 Varieties Total 590.747 Anova shows there is significant difference in cotyledon leafwidth in 20-day-old seedlings.

TABLE 4 Length of 4^(th) leaf (cm) Red Crispita Haflex Telex (LS16037)6.5 7 6.2 6 6.3 7.5 6 7 6.5 6 7.2 7.9 7.4 7 6.5 5 7 8 6.5 8 7.9 7.9 7.57.3 5.5 7.5 7 5.5 7.5 6.5 6.3 7 6.5 6.5 6.8 7.6 7.5 7.2 7.5 5 6.7 7.34.3 9 7.5 5.7 7.8 6.5 6 6.5 7 6.5 7.5 7.6 6.6 8.3 7.2 7.8 7.4 7.9 ANOVA:Single factor Summary Groups Count Sum Average Variance Halfex 20 124.56.225 0.894 Telex 20 146.2 7.31 0.396 Red Crispita 20 143.9 7.20 0.329ANOVA F critical Source DF SS MS F Prob > F (2,57) Between 2 14.21 7.10513.1715 <.0001 3.158 Varieties Within 57 30.75 0.539 Varieties Total 5944.95 Anova shows there is significant difference in 4^(th) leaf length.

TABLE 5 Width of 4^(th) leaf (cm) Red Crispita Haflex Telex (LS16037)4.8 3.5 4.5 3.7 4.5 4.5 4 4.6 4.7 4.5 4 5 4.7 3.5 3.5 3.5 4.3 4 4 4 4.45 3.2 4.3 3.7 4.5 4.4 3.5 4 4.5 2.5 3.5 4 4 5 4.4 4 4.7 4.5 3.5 4.7 4.74 3.9 4.3 3.7 4 4.6 5.5 5.4 4.6 3 5.4 4.5 4 4.5 4.5 4.5 5 3.5 ANOVA:Single factor Summary Groups Count Sum Average Variance Halfex 20 80.14.01 0.482 Telex 20 86.2 4.31 0.399 Red Crispita 20 87.4 4.37 0.138ANOVA F critical Source DF SS MS F Prob > F (2,57) Between 2 1.532 0.7662.26 0.1139 3.158 Varieties Within 57 19.35 0.34 Varieties Total 5920.88 Anova shows there is no significant difference in width of the4^(th) leaf.

Further Embodiments of the Invention

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign nucleic acids including additional or modified versions ofnative (endogenous) nucleic acids (optionally driven by a non-nativepromoter) in order to alter the traits of a plant in a specific manner.Any nucleic acid sequences, whether from a different species or from thesame species, which are introduced into the genome using transformationor various breeding methods, are referred to herein collectively as“transgenes.” Over the last fifteen to twenty years, several methods forproducing transgenic plants have been developed, and in particularembodiments the present invention also relates to transformed versionsof lettuce plants disclosed herein.

Genetic engineering techniques can be used (alone or in combination withbreeding methods) to introduce one or more desired added traits intoplant, for example, lettuce cultivar Red Crispita or progeny or lettuceplants derived thereof.

Plant transformation generally involves the construction of anexpression vector that will function in plant cells. Optionally, such avector comprises one or more nucleic acids comprising a coding sequencefor a polypeptide or an untranslated functional RNA under control of, oroperatively linked to, a regulatory element (for example, a promoter).In representative embodiments, the vector(s) may be in the form of aplasmid, and can be used alone or in combination with other plasmids, toprovide transformed lettuce plants using transformation methods asdescribed herein to incorporate transgenes into the genetic material ofthe lettuce plant.

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct nucleic acid transfermethod, such as microprojectile-mediated delivery (e.g., with abiolistic device), DNA injection, Agrobacterium-mediated transformation,electroporation, and the like. Transformed plants obtained from theplants (and parts and tissue culture thereof) of the invention areintended to be within the scope of this invention.

Expression Vectors for Plant Transformation—Selectable markers.

Expression vectors typically include at least one nucleic acidcomprising or encoding a selectable marker, operably linked to aregulatory element (for example, a promoter) that allows transformedcells containing the marker to be either recovered by negativeselection, e.g., inhibiting growth of cells that do not contain theselectable marker, or by positive selection, e.g., screening for theproduct encoded by the selectable marker. Many commonly used selectablemarkers for plant transformation are well known in the transformationart, and include, for example, nucleic acids that code for enzymes thatmetabolically detoxify a selective chemical agent which may be anantibiotic or an herbicide, or nucleic acids that encode an alteredtarget which is insensitive to the inhibitor. Positive selection methodsare also known in the art.

One commonly used selectable marker for plant transformation is aneomycin phosphotransferase II (nptII) coding sequence, for example,isolated from transposon Tn5, which when placed under the control ofplant regulatory signals confers resistance to kanamycin. Fraley, etal., PNAS, 80:4803 (1983). Another commonly used selectable marker ishygromycin phosphotransferase, which confers resistance to theantibiotic hygromycin. Vanden Elzen, et al., Plant Mol. Biol., 5:299(1985).

Additional selectable markers of bacterial origin that confer resistanceto antibiotics include gentamycin acetyl transferase, streptomycinphosphotransferase, aminoglycoside-3′-adenyl transferase, the bleomycinresistance determinant. Hayford, et al., Plant Physiol., 86:1216 (1988);Jones, et al., Mol. Gen. Genet., 210:86 (1987); Svab, et al., Plant Mol.Biol., 14:197 (1990); Hille, et al., Plant Mol. Biol., 7:171 (1986).Other selectable markers confer resistance to herbicides such asglyphosate, glufosinate, or bromoxynil. Comai, et al., Nature,317:741-744 (1985); Gordon-Kamm, et al., Plant Cell, 2:603-618 (1990);and Stalker, et al., Science, 242:419-423 (1988).

Selectable markers for plant transformation that are not of bacterialorigin include, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase, and plant acetolactatesynthase. Eichholtz, et al., Somatic Cell Mol. Genet., 13:67 (1987);Shah, et al., Science, 233:478 (1986); and Charest, et al., Plant CellRep., 8:643 (1990).

Another class of selectable marker for plant transformation involvesscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These selectable markers areparticularly useful to quantify or visualize the spatial pattern ofexpression of a transgene in specific tissues and are frequentlyreferred to as a reporter gene because they can be fused to transgene orregulatory sequence for the investigation of nucleic acid expression.Commonly used reporters for screening presumptively transformed cellsinclude alpha-glucuronidase (GUS), alpha-galactosidase, luciferase andchloramphenicol, acetyltransferase. Jefferson, R. A., Plant Mol. Biol.,5:387 (1987); Teeri, et al., EMBO J., 8:343 (1989); Koncz, et al., PNAS,84:131 (1987); and DeBlock, et al., EMBO J., 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissues are available. Molecular Probes,Publication 2908, IMAGENE GREEN, pp. 1-4 (1993) and Naleway, et al., J.Cell Biol., 115:151a (1991).

Green Fluorescent Protein (GFP) is also utilized as a marker for nucleicacid expression in prokaryotic and eukaryotic cells. Chalfie, et al.,Science, 263:802 (1994). GFP and mutants of GFP may be used asscreenable markers.

Expression Vectors for Plant Transformation—Promoters.

Transgenes included in expression vectors are generally driven by anucleotide sequence comprising a regulatory element (for example, apromoter). Numerous types of promoters are well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells.

Examples of promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues, such asleaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma.Such promoters are referred to as “tissue-preferred.” Promoters thatinitiate transcription only in certain tissue are referred to as“tissue-specific.” A “cell type” specific promoter preferentially drivesexpression in certain cell types in one or more organs, for example,vascular cells in roots or leaves. An “inducible” promoter is a promoterthat is under environmental control. Examples of environmentalconditions that may affect transcription by inducible promoters includeanaerobic conditions or the presence of light. Tissue-specific,tissue-preferred, cell type specific, and inducible promoters constitutethe class of “non-constitutive” promoters. A “constitutive” promoter isa promoter that is active under most environmental conditions.

A. Inducible Promoters:

An inducible promoter is operably linked to a nucleic acid forexpression in a plant. Optionally, the inducible promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a nucleic acid for expression in the plant. With aninducible promoter, the rate of transcription increases in response toan inducing agent.

Any inducible promoter can be used in the instant invention. See Ward,et al., Plant Mol. Biol., 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Melt, et al., PNAS, 90:4567-4571 (1993));promoter from the In2 gene from maize which responds tobenzenesulfonamide herbicide safeners (Hershey, et al., Mol. Gen.Genet., 227:229-237 (1991) and Gatz, et al., Mol. Gen. Genet., 243:32-38(1994)) or Tet repressor from Tn10 (Gatz, et al., Mol. Gen. Genet.,227:229-237 (1991)). A representative inducible promoter is a promoterthat responds to an inducing agent to which plants do not normallyrespond. An exemplary inducible promoter is the inducible promoter froma steroid hormone gene, the transcriptional activity of which is inducedby a glucocorticosteroid hormone. Schena, et al., PNAS, 88:0421 (1991).

B. Constitutive Promoters:

A constitutive promoter is operably linked to a nucleic acid forexpression in a plant or the constitutive promoter is operably linked toa nucleotide sequence encoding a signal sequence which is operablylinked to a nucleic acid for expression in a plant.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell, et al., Nature, 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy, et al., Plant Cell, 2:163-171 (1990));ubiquitin (Christensen, et al., Plant Mol. Biol., 12:619-632 (1989) andChristensen, et al., Plant Mol. Biol., 18:675-689 (1992)); pEMU (Last,et al., Theor. Appl. Genet., 81:581-588 (1991)); MAS (Velten, et al.,EMBO J., 3:2723-2730 (1984)) and maize H3 histone (Lepetit, et al., Mol.Gen. Genet., 231:276-285 (1992) and Atanassova, et al., Plant J., 2(3):291-300 (1992)). The ALS promoter, Xbal/Ncol fragment 5′ to theBrassica napus ALS3 structural gene (or a nucleotide sequence similarityto said Xbal/Ncol fragment), represents a particularly usefulconstitutive promoter. See PCT Application No. WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters:

A tissue-specific promoter is operably linked to a nucleic acid forexpression in a plant. Optionally, the tissue-specific promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a nucleic acid for expression in a plant.Plants transformed with a nucleic acid of interest operably linked to atissue-specific promoter transcribe the nucleic acid of interestexclusively, or preferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai, et al., Science, 23:476-482(1983) and Sengupta-Gopalan, et al., PNAS, 82:3320-3324 (1985)); aleaf-specific and light-induced promoter such as that from cab orrubisco (Simpson, et al., EMBO J., 4(11):2723-2729 (1985) and Timko, etal., Nature, 318:579-582 (1985)); an anther-specific promoter such asthat from LAT52 (Twell, et al., Mol. Gen. Genet., 217:240-245 (1989)); apollen-specific promoter such as that from Zm13 (Guerrero, et al., Mol.Gen. Genet., 244:161-168 (1993)) or a microspore-preferred promoter suchas that from apg (Twell, et al., Sex. Plant Reprod., 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments.

Transport of polypeptides produced by transgenes to a subcellularcompartment such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall, or mitochondrion, or for secretion into the apoplast, isgenerally accomplished by means of operably linking a nucleotidesequence encoding a signal sequence to the 5′ and/or 3′ region of anucleic acid encoding the polypeptide of interest. Signal sequences atthe 5′ and/or 3′ end of the coding sequence target the polypeptide toparticular subcellular compartments.

The presence of a signal sequence can direct a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker, et al., Plant Mol. Biol., 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley,” Plant Mol. Biol., 9:3-17 (1987); Lerner, et al., PlantPhysiol., 91:124-129 (1989); Fontes, et al., Plant Cell, 3:483-496(1991); Matsuoka, et al., PNAS, 88:834 (1991); Gould, et al., J. Cell.Biol., 108:1657 (1989); Creissen, et al., Plant J, 2:129 (1991);Kalderon, et al., A short amino acid sequence able to specify nuclearlocation, Cell, 39:499-509 (1984); and Steifel, et al., Expression of amaize cell wall hydroxyproline-rich glycoprotein gene in early leaf androot vascular differentiation, Plant Cell, 2:785-793 (1990).

Foreign Polypeptide Transgenes and Agronomic Transgenes.

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign polypeptide then canbe extracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981).

According to a representative embodiment, the transgenic plant providedfor commercial production of foreign protein is a lettuce plant of theinvention. In another embodiment, the biomass of interest is seed. Forthe relatively small number of transgenic plants that show higher levelsof expression, a genetic map can be generated, for example viaconventional RFLP, PCR, and SSR analysis, which identifies theapproximate chromosomal location of the integrated DNA molecule. Forexemplary methodologies in this regard, see Methods in Plant MolecularBiology and Biotechnology, Glick and Thompson Eds., 269:284, CRC Press,Boca Raton (1993). Map information concerning chromosomal location isuseful for proprietary protection of a subject transgenic plant. Ifunauthorized propagation is undertaken and crosses made with othergermplasm, the map of the integration region can be compared to similarmaps for suspect plants, to determine if the latter have a commonparentage with the subject plant. Map comparisons can involvehybridizations, RFLP, PCR, SSR, and sequencing, all of which areconventional techniques.

Likewise, by means of the present invention, agronomic transgenes andother desired added traits can be expressed in transformed plants (andtheir progeny, e.g., produced by breeding methods). More particularly,plants can be genetically engineered to express various phenotypes ofagronomic interest or other desired added traits. Exemplary nucleicacids of interest in this regard conferring a desired added trait(s)include, but are not limited to, those categorized below:

A. Transgenes that Confer Resistance to Pests or Disease:

1. Plant disease resistance transgenes. Plant defenses are oftenactivated by specific interaction between the product of a diseaseresistance gene (R) in the plant and the product of a correspondingavirulence (Avr) gene in the pathogen. A plant line can be transformedwith a cloned resistance transgene to engineer plants that are resistantto specific pathogen strains. See, for example, Jones, et al., Science,266:789 (1994) (cloning of the tomato Cf-9 gene for resistance toCladosporium fulvum); Martin, et al., Science, 262:1432 (1993) (tomatoPto gene for resistance to Pseudomonas syringae pv. tomato encodes aprotein kinase); and Mindrinos, et al., Cell, 78:1089 (1994)(Arabidopsis RSP2 gene for resistance to Pseudomonas syringae).

2. A Bacillus thuringiensis protein, a derivative thereof, or asynthetic polypeptide modeled thereon. See, for example, Geiser, et al.,Gene, 48:109 (1986), who disclose the cloning and nucleotide sequence ofa Bt delta-endotoxin gene. Moreover, DNA molecules encodingdelta-endotoxin transgenes can be purchased from American Type CultureCollection, Manassas, Va., for example, under ATCC Accession Nos. 40098,67136, 31995, and 31998.

3. A lectin. See, for example, the disclosure by Van Damme, et al.,Plant Mol. Biol., 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin transgenes.

4. A vitamin-binding protein such as avidin. See, e.g., PCT ApplicationNo. US 93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

5. An enzyme inhibitor, for example, a protease or proteinase inhibitor,or an amylase inhibitor. See, for example, Abe, et al., J. Biol. Chem.,262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor); Huub, et al., Plant Mol. Biol., 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); and Sumitani,et al., Biosci. Biotech. Biochem., 57:1243 (1993) (nucleotide sequenceof Streptomyces nitrosporeus alpha-amylase inhibitor).

6. An insect-specific hormone or pheromone, such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., Nature, 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

7. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem., 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor) and Pratt, etal., Biochem. Biophys. Res. Comm., 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also, U.S. Pat. No. 5,266,317 toTomalski, et al., who disclose transgenes encoding insect-specific,paralytic neurotoxins.

8. An insect-specific venom produced in nature, by a snake, a wasp, etc.For example, see Pang, et al, Gene, 116:165 (1992), for disclosure ofheterologous expression in plants of a transgene coding for a scorpioninsectotoxic peptide.

9. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative,or another non-protein molecule with insecticidal activity.

10. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, and a glucanase, whether natural or synthetic. See PCTApplication No. WO 93/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase transgene. DNA moleculeswhich contain chitinase-encoding sequences can be obtained, for example,from the ATCC under Accession Nos. 39637 and 67152. See also, Kramer, etal., Insect Biochem. Mol. Biol., 23:691 (1993), who teach the nucleotidesequence of a cDNA encoding tobacco hornworm chitinase, and Kawalleck,et al., Plant Mol. Biol., 21:673 (1993), who provide the nucleotidesequence of the parsley ubi4-2 polyubiquitin transgene.

11. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., Plant Mol. Biol., 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess,et al., Plant Physiol., 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

12. A hydrophobic moment peptide. See PCT Application No. WO 95/16776(disclosure of peptide derivatives of tachyplesin which inhibit fungalplant pathogens) and PCT Application No. WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance).

13. A membrane permease, a channel former, or a channel blocker. Forexample, see the disclosure of Jaynes, et al., Plant Sci., 89:43 (1993),of heterologous expression of a cecropin-beta, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

14. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein transgene is derived,as well as by related viruses. See Beachy, et al., Ann. Rev.Phytopathol., 28:451 (1990). Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus, and tobacco mosaic virus. Id.

15. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. SeeTaylor, et al., Abstract #497, Seventh Intl Symposium on MolecularPlant-Microbe Interactions, Edinburgh, Scotland (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

16. A virus-specific antibody. See, for example, Tavladoraki, et al.,Nature, 366:469 (1993), who show that transgenic plants expressingrecombinant antibody transgenes are protected from virus attack.

17. A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo-alpha-1,4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient released bysolubilizing plant cell wall homo-alpha-1,4-D-galacturonase. See Lamb,et al., Bio/technology, 10:1436 (1992). The cloning and characterizationof a transgene which encodes a bean endopolygalacturonase-inhibitingprotein is described by Toubart, et al., Plant J., 2:367 (1992).

18. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., Bio/technology, 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivatingtransgene have an increased resistance to fungal disease.

19. A lettuce mosaic potyvirus (LMV) coat protein transgene introducedinto Lactuca sativa in order to increase its resistance to LMVinfection. See Dinant, et al., Mol. Breeding, 3:1, 75-86 (1997).

Any disease or present resistance transgenes, including thoseexemplified above, can be introduced into a lettuce plant of theinvention through a variety of means including but not limited totransformation and breeding.

B. Transgenes that Confer Resistance to an Herbicide:

Exemplary polynucleotides encoding polypeptides that confer traitsdesirable for herbicide resistance include acetolactate synthase (ALS)mutants that lead to herbicide resistance such as the S4 and/or Hramutations ((resistance to herbicides including sulfonylureas,imidazolinones, triazolopyrimidines, pyrimidinyl thiobenzoates);glyphosate resistance (e.g.,5-enol-pyrovyl-shikimate-3-phosphate-synthase (EPSPS) transgene,including but not limited to those described in U.S. Pat. Nos.4,940,935, 5,188,642, 5,633,435, 6,566,587, 7,674,598 as well as allrelated application; or the glyphosate N-acetyltransferase (GAT)transgene, described in Castle et al., Science, 2004, 304:1151-1154; andin U.S. Patent Application Publication Nos. 20070004912, 20050246798,and 20050060767)); glufosinate resistance (e.g., BAR; see e.g., U.S.Pat. No. 5,561,236); 2,4-D resistance (e.g., aryloxy alkanoatedioxygenase or AAD-1, AAD-12, or AAD-13), HPPD resistance (e.g.,Pseudomonas HPPD) and PPO resistance (e.g., fomesafen,acifluorfen-sodium, oxyfluorfen, lactofen, fluthiacet-methyl,saflufenacil, flumioxazin, flumiclorac-pentyl, carfentrazone-ethyl,sulfentrazone,); a cytochrome P450 or variant thereof that confersherbicide resistance or tolerance to, inter alia, HPPD-inhibitingherbicides, PPO-inhibiting herbicides and ALS-inhibiting herbicides(U.S. Patent Application Publication No. 20090011936; U.S. Pat. Nos.6,380,465; 6,121,512; 5,349,127; 6,649,814; and 6,300,544; and PCTInternational Publication No. WO 2007/000077); dicamba resistance (e.g.,dicamba monoxygenase), and traits desirable for processing or processproducts such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils(e.g., fatty acid desaturase transgenes (U.S. Pat. No. 5,952,544; PCTInternational Publication No. WO 94/11516)); modified starches (e.g.,ADPG pyrophosphorylases (AGPase), starch synthases (SS), starchbranching enzymes (SBE), and starch debranching enzymes (SDBE)); andpolymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoAreductase (Schubert et al., J. Bacteriol., 1988, 170:5837-5847)facilitate expression of polyhydroxyalkanoates (PHAs)).

In embodiments, the polynucleotide encodes a polypeptide conferringresistance to an herbicide selected from glyphosate, sulfonylurea,imidazolinone, dicamba, glufosinate, phenoxy proprionic acid,L-phosphinothricin, cyclohexone, cyclohexanedione, triazine, andbenzonitrile.

Any transgene conferring herbicide resistance, including thoseexemplified above, can be introduced into the lettuce plants of theinvention through a variety of means including, but not limited to,transformation (e.g., genetic engineering techniques) and crossing.

C. Transgenes that Confer or Contribute to a Value-Added Trait:

1. Increased iron content of the lettuce, for example, by introducinginto a plant a soybean ferritin transgene as described in Goto, et al.,Acta Horticulturae., 521, 101-109 (2000).

2. Decreased nitrate content of leaves, for example, by introducing intoa lettuce a transgene coding for a nitrate reductase. See, for example,Curtis, et al., Plant Cell Rep., 18:11, 889-896 (1999).

3. Increased sweetness of the lettuce by introducing a transgene codingfor monellin that elicits a flavor 100,000 times sweeter than sugar on amolar basis. See Penarrubia, et al., Bio/technology, 10:561-564 (1992).

4. Modified fatty acid metabolism, for example, by introducing into aplant an antisense sequence directed against stearyl-ACP desaturase toincrease stearic acid content of the plant. See Knultzon, et al., PNAS,89:2625 (1992).

5. Modified carbohydrate composition effected, for example, byintroducing into plants a transgene coding for an enzyme that alters thebranching pattern of starch. See Shiroza, et al., J. Bacteria, 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase transgene); Steinmetz, et al., Mol. Gen. Genet.,20:220 (1985) (nucleotide sequence of Bacillus subtilis levansucrasetransgene); Pen, et al., Bio/technology, 10:292 (1992) (production oftransgenic plants that express Bacillus lichenifonnis alpha-amylase);Elliot, et al., Plant Mol. Biol., 21:515 (1993) (nucleotide sequences oftomato invertase transgenes); Sogaard, et al., J. Biol. Chem., 268:22480(1993) (site-directed mutagenesis of barley alpha-amylase transgene);and Fisher, et al., Plant Physiol., 102:1045 (1993) (maize endospermstarch branching enzyme II).

Any transgene that confers or contributes a value-added trait, includingthose exemplified above, can be introduced into the lettuce plants ofthe invention through a variety of means including, but not limited to,transformation (e.g., genetic engineering techniques) and crossing.

D. Transgenes that Control Male-Sterility:

1. Introduction of a deacetylase transgene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See, e.g., International Publication WO 01/29237.

2. Introduction of various stamen-specific promoters. See, e.g.,International Publications WO 92/13956 and WO 92/13957.

3. Introduction of the barnase and the barstar transgenes. See, e.g.,Paul, et al., Plant Mol. Biol., 19:611-622 (1992).

Any transgene that controls male sterility, including those exemplifiedabove, can be introduced into the lettuce plants of the inventionthrough a variety of means including, but not limited to, transformation(e.g., genetic engineering techniques) and crossing.

Methods for Plant Transformation.

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A. Agrobacterium-Mediated Transformation.

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch, et al., Science, 227:1229 (1985); Curtis, et al., Journal ofExperimental Botany, 45:279, 1441-1449 (1994); Torres, et al., PlantCell Tissue and Organ Culture, 34:3, 279-285 (1993); and Dinant, et al.,Molecular Breeding, 3:1, 75-86 (1997). A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci., 10:1(1991). Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated transgene transfer are provided by Gruber, etal., supra, Miki, et al., supra, and Moloney, et al., Plant Cell Rep.,8:238 (1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Transgene Transfer.

Several methods of plant transformation collectively referred to asdirect transgene transfer have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 micron to4 micron. The expression vector is introduced into plant tissues with abiolistic device that accelerates the microprojectiles to speeds of 300m/s to 600 m/s which is sufficient to penetrate plant cell walls andmembranes. Russell, D. R., et al., Plant Cell Rep., 12 (3, January),165-169 (1993); Aragao, F. J. L., et al., Plant Mol. Biol., 20 (2,October), 357-359 (1992); Aragao, F. J. L., et al., Plant Cell Rep., 12(9, July), 483-490 (1993); Aragao, Theor. Appl. Genet., 93:142-150(1996); Kim, J., Minamikawa, T., Plant Sci., 117:131-138 (1996);Sanford, et al., Part. Sci. Technol., 5:27 (1987); Sanford, J. C.,Trends Biotech., 6:299 (1988); Klein, et al., Bio/technology, 6:559-563(1988); Sanford, J. C., Physiol. Plant, 7:206 (1990); Klein, et al.,Bio/technology, 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang, et al., Bio/technology, 9:996 (1991).Alternatively, liposome and spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes, et al., EMBO J.,4:2731 (1985) and Christou, et al., PNAS, 84:3962 (1987). Direct uptakeof DNA into protoplasts using CaCl.sub.2 precipitation, polyvinylalcohol, or poly-L-ornithine has also been reported. Hain, et al., Mol.Gen. Genet., 199:161 (1985) and Draper, et al., Plant Cell Physiol.,23:451 (1982). Electroporation of protoplasts and whole cells andtissues have also been described. Saker, M., Kuhne, T., BiologiaPlantarum, 40(4):507-514 (1997/98); Donn, et al., In Abstracts of VllthInternational Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p.53 (1990); D'Halluin, et al., Plant Cell, 4:1495-1505 (1992); andSpencer, et al., Plant Mol. Biol., 24:51-61 (1994). See also Chupean, etal., Bio/technology, 7:5, 503-508 (1989).

Following transformation of plant target tissues, expression of theabove-described selectable marker transgenes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic lettuce line. The transgenic lettuce line couldthen be crossed with another (non-transformed or transformed) line inorder to produce a new transgenic lettuce line. Alternatively, a genetictrait that has been engineered into a particular plant cultivar usingthe foregoing transformation techniques could be introduced into anotherline using traditional breeding (e.g., backcrossing) techniques that arewell known in the plant breeding arts. For example, a backcrossingapproach could be used to move an engineered trait from a public,non-elite inbred line into an elite inbred line, or from an inbred linecontaining a foreign transgene in its genome into an inbred line orlines which do not contain that transgene. As used herein, “crossing”can refer to a simple X by Y cross, or the process of backcrossing,depending on the context.

Gene Conversions.

When the term “lettuce plant” is used in the context of the presentinvention, this term also includes any gene conversions of that plant orvariety. The term “gene converted plant” as used herein refers to thoselettuce plants that are developed, for example, by backcrossing, geneticengineering and/or mutation, wherein essentially all of the desiredmorphological and physiological characteristics of a variety arerecovered in addition to the one or more genes transferred into thevariety. To illustrate, backcrossing methods can be used with thepresent invention to improve or introduce a characteristic into thevariety. The term “backcrossing” as used herein refers to the repeatedcrossing of a hybrid progeny back to the recurrent parent, e.g.,backcrossing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times to the recurrentparent. The parental plant that contributes the gene for the desiredcharacteristic is termed the “nonrecurrent” or “donor parent.” Thisterminology refers to the fact that the nonrecurrent parent is generallyused one time in the breeding e.g., backcross) protocol and thereforedoes not recur. The gene that is transferred can be a native gene, amutated native gene or a transgene introduced by genetic engineeringtechniques into the plant (or ancestor thereof). The parental plant intowhich the gene(s) from the nonrecurrent parent are transferred is knownas the “recurrent” parent as it is used for multiple rounds in thebackcrossing protocol. Poehlman & Sleper (1994) and Fehr (1993). In atypical backcross protocol, the original variety of interest (recurrentparent) is crossed to a second variety (nonrecurrent parent) thatcarries the gene(s) of interest to be transferred. The resulting progenyfrom this cross are then crossed again to the recurrent parent and theprocess is repeated until a plant is obtained wherein essentially all ofthe desired morphological and physiological characteristics of therecurrent parent are recovered in the converted plant in addition to thetransferred gene(s) and associated trait(s) from the nonrecurrentparent.

Many gene traits have been identified that are not regularly selected inthe development of a new line but that can be improved by backcrossingtechniques. Gene traits may or may not be transgenic. Examples of thesetraits include, but are not limited to, male sterility, modified fattyacid metabolism, modified carbohydrate metabolism, herbicide resistance,pest or disease resistance (e.g., resistance to bacterial, fungal, orviral disease), insect resistance, enhanced nutritional quality,increased sweetness, increased flavor, improved ripening control,improved salt tolerance, industrial usage, yield stability, and yieldenhancement. These genes are generally inherited through the nucleus.

Tissue Culture.

Further reproduction of lettuce plants variety can occur by tissueculture and regeneration. Tissue culture of various tissues of lettuceand regeneration of plants therefrom is well known and widely published.For example, reference may be had to Teng, et al., HortScience, 27:9,1030-1032 (1992); Teng, et al., HortScience, 28:6, 669-1671 (1993);Zhang, et al., Journal of Genetics and Breeding, 46:3, 287-290 (1992);Webb, et al., Plant Cell Tissue and Organ Culture, 38:1, 77-79 (1994);Curtis, et al., Journal of Experimental Botany, 45:279, 1441-1449(1994); Nagata, et al., Journal for the American Society forHorticultural Science, 125:6, 669-672 (2000); and Ibrahim, et al., PlantCell Tissue and Organ Culture, 28(2), 139-145 (1992). It is clear fromthe literature that the state of the art is such that these methods ofobtaining plants are routinely used and have a very high rate ofsuccess. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce lettuce plants havingdesired characteristics of lettuce cultivar Red Crispita. Optionally,lettuce plants can be regenerated from the tissue culture of theinvention comprising all or essentially all of the physiological andmorphological characteristics of lettuce cultivar Red Crispita.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, suckers, and the like. Meansfor preparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and5,977,445 describe certain techniques.

Additional Breeding Methods.

This invention is also directed to methods for producing a lettuce plantby crossing a first parent lettuce plant with a second parent lettuceplant wherein the first or second parent lettuce plant is a plant oflettuce cultivar Red Crispita. Further, both first and second parentlettuce can come from lettuce cultivar Red Crispita. Thus, any of thefollowing exemplary methods using lettuce cultivar Red Crispita are partof this invention: selfing, backcrosses, hybrid production, crosses topopulations, double haploid production, and the like. All plantsproduced using lettuce cultivar Red Crispita as at least one parent arewithin the scope of this invention, including those developed fromlettuce plants derived from lettuce cultivar Red Crispita.Advantageously, lettuce cultivar Red Crispita can be used in crosseswith other, different, lettuce plants to produce the first generation(F₁) lettuce hybrid seeds and plants with desirable characteristics. Thelettuce plants of the invention can also be used for transformationwhere exogenous transgenes are introduced and expressed by the plants ofthe invention. Genetic variants created either through traditionalbreeding methods or through transformation of the cultivars of theinvention by any of a number of protocols known to those of skill in theart are intended to be within the scope of this invention.

The following describes exemplary breeding methods that may be used withlettuce cultivar Red Crispita in the development of further lettuceplants. One such embodiment is a method for developing lettuce cultivarRed Crispita progeny lettuce plants in a lettuce plant breeding programcomprising: obtaining a plant, or a part thereof, of lettuce cultivarRed Crispita, utilizing said plant or plant part as a source of breedingmaterial, and selecting a lettuce cultivar Red Crispita progeny plantwith molecular markers in common with lettuce cultivar Red Crispitaand/or with some, all or essentially all of the morphological and/orphysiological characteristics of lettuce cultivar Red Crispita (see,e.g., Table 1 and/or Tables 2 to 5). In representative embodiments, theprogeny plant has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of themorphological and physiological characteristics of lettuce cultivar RedCrispita (e.g., as described in Table 1 and/or Tables 2 to 5, such asred mature leaf color, thick and crisp leaves, and/or high resistance toBremia lactucae races 1-18 and 20-32), or even all of the morphologicaland physiological characteristics of lettuce cultivar Red Crispita sothat said progeny lettuce plant is not significantly different for saidtraits than lettuce cultivar Red Crispita, as determined at the 5%significance level when grown in the same environmental conditions;optionally, with the presence of one or more desired additional traits(e.g., male sterility, disease resistance, pest or insect resistance,herbicide resistance, and the like). Breeding steps that may be used inthe breeding program include pedigree breeding, backcrossing, mutationbreeding and/or recurrent selection. In conjunction with these steps,techniques such as RFLP-enhanced selection, genetic marker enhancedselection (for example, SSR markers) and/or and the making of doublehaploids may be utilized.

Another representative method involves producing a population of lettucecultivar Red Crispita progeny plants, comprising crossing lettucecultivar Red Crispita with another lettuce plant, thereby producing apopulation of lettuce plants that, on average, derives at least 6.25%,12.5%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% of its alleles (i.e., TAC) from lettucecultivar Red Crispita, e.g., at least about 6.25%, 12.5%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% of the genetic complement of lettuce cultivar Red Crispita. Oneembodiment of this invention is the lettuce plant produced by thismethod and that has obtained at least 6.25%, 12.5%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%of its alleles from lettuce cultivar Red Crispita, and optionally is theresult of a breeding process comprising one or two breeding crosses andone or more of selfing, sibbing, backcrossing and/or double haploidtechniques in any combination and any order. In embodiments, thebreeding process does not include a breeding cross, and comprisesselfing, sibbing, backcrossing and or double haploid technology. A plantof this population may be selected and repeatedly selfed or sibbed witha lettuce plant resulting from these successive filial generations.Another approach is to make double haploid plants to achievehomozygosity.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, pp. 261-286 (1987). In embodiments, the inventionencompasses Red Crispita progeny plants having a combination of at least2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the characteristics as describedherein for lettuce cultivar Red Crispita, so that said progeny lettuceplant is not significantly different for said traits than lettucecultivar Red Crispita, as determined at the 5% significance level whengrown in the same environmental conditions. Using techniques describedherein and those known in the art, molecular markers may be used toidentify said progeny plant as progeny of lettuce cultivar Red Crispita.Mean trait values may be used to determine whether trait differences aresignificant, and optionally the traits are measured on plants grownunder the same environmental conditions.

Progeny of lettuce cultivar Red Crispita may also be characterizedthrough their filial relationship with lettuce cultivar Red Crispita, asfor example, being within a certain number of breeding crosses oflettuce cultivar Red Crispita. A breeding cross is a cross made tointroduce new genetics into the progeny, and is distinguished from across, such as a self or a sib cross or a backcross to Red Crispita as arecurrent parent, made to select among existing genetic alleles. Thelower the number of breeding crosses in the pedigree, the closer therelationship between lettuce cultivar Red Crispita and its progeny. Forexample, progeny produced by the methods described herein may be within1, 2, 3, 4, 5 or more breeding crosses of lettuce cultivar Red Crispita.

In representative embodiments, a lettuce plant derived from lettucecultivar Red Crispita comprises cells comprising at least one set ofchromosomes derived from lettuce cultivar Red Crispita. In embodiments,the lettuce plant or population of lettuce plants derived from lettucecultivar Red Crispita comprises, on average, at least 6.25%, 12.5%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% of its alleles (i.e., TAC) from lettuce cultivar RedCrispita, e.g., at least about 6.25%, 12.5%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of thegenetic complement of lettuce cultivar Red Crispita, and optionally isthe result of a breeding process comprising one or two breeding crossesand one or more of selfing, sibbing, backcrossing and/or double haploidtechniques in any combination and any order. In embodiments, thebreeding process does not include a breeding cross, and comprisesselfing, sibbing, backcrossing and or double haploid technology. Inembodiments, the lettuce plant derived from lettuce cultivar RedCrispita is one, two, three, four, five or more breeding crosses removedfrom lettuce cultivar Red Crispita.

In representative embodiments, a plant derived from lettuce cultivar RedCrispita is a double haploid plant, a hybrid plant or an inbred plant.

In embodiments, a hybrid or derived plant from lettuce cultivar RedCrispita comprises a desired added trait. In representative embodiments,a lettuce plant derived from lettuce cultivar Red Crispita comprises allof the morphological and physiological characteristics of lettucecultivar Red Crispita (e.g., as described in Table 1 and/or Tables 2 to5, for example, red mature leaf color, thick and crisp leaves, and highresistance to Bremia lactucae races 1-18 and 20-32). In embodiments, thelettuce plant derived from lettuce cultivar Red Crispita comprisesessentially all of the morphological and physiological characteristicsof lettuce cultivar Red Crispita (e.g., as described in Table 1 and/orTables 2 to 5, for example, red mature leaf color, thick and crispleaves, and high resistance to Bremia lactucae races 1-18 and 20-32),with the addition of a desired added trait.

Those skilled in the art will appreciate that any of the traitsdescribed above with respect to plant transformation methods can beintroduced into a plant of the invention (e.g., lettuce cultivar RedCrispita and hybrid lettuce plants and other lettuce plants derivedtherefrom) using breeding techniques.

Genetic Analysis of Lettuce Cultivar Red Crispita.

The invention further provides a method of determining a geneticcharacteristic of lettuce cultivar Red Crispita or a progeny thereof,e.g., a method of determining a genotype of lettuce cultivar RedCrispita or a progeny thereof. In embodiments, the method comprisesdetecting in the genome of a Red Crispita plant, or a progeny plantthereof, at least a first polymorphism (e.g., using nucleic acidamplification, nucleic acid sequencing and/or one or more molecularmarkers). To illustrate, in embodiments, the method comprises obtaininga sample of nucleic acids from the plant and detecting at least a firstpolymorphism in the nucleic acid sample. Optionally, the method maycomprise detecting a plurality of polymorphisms (e.g., two or more,three or more, four or more, five or more, six or more, eight or more orten or more polymorphisms, etc.) in the genome of the plant. Inrepresentative embodiments, the method further comprises storing theresults of the step of detecting the polymorphism(s) on a computerreadable medium. The invention further provides a computer readablemedium produced by such a method.

DEPOSIT INFORMATION

Applicants have made a deposit of at least 2500 seeds of lettucecultivar Red Crispita with the American Type Culture Collection (ATCC),10801 University Boulevard, Manassas, Va., 20110-2209 U.S.A. under ATCCDeposit No. ______ on ______. This deposit of lettuce variety RedCrispita will be maintained in the ATCC depository, which is a publicdepository, for a period of 30 years, or 5 years after the most recentrequest, or for the effective life of the patent, whichever is longer,and will be replaced if any of the deposited seed becomes nonviableduring that period. Additionally, Applicants have satisfied all therequirements of 37 C.F.R. §§ 1.801-1.809, including providing anindication of the viability of the samples. Access to this deposit willbe made available during the pendency of this application to theCommissioner upon request. Upon the issuance of a patent on the variety,the variety will be irrevocably and without restriction released to thepublic by providing access to the deposit of at least 2500 seeds of thevariety with the ATCC. Applicants impose no restrictions on theavailability of the deposited material from the ATCC; however,Applicants have no authority to waive any restrictions imposed by law onthe transfer of biological material or its transportation in commerce.Applicants do not waive any infringement of its rights granted underthis patent or under the Plant Variety Protection Act (7 USC § 2321 etseq.).

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding.However, it will be apparent that certain changes and modifications suchas single gene modifications and mutations, somaclonal variants, variantindividuals selected from large populations of the plants of the instantinbred and the like may be practiced within the scope of the invention.

What is claimed is:
 1. A seed of lettuce cultivar Red Crispita, arepresentative sample of seed having been deposited under ATCC AccessionNo. ______.
 2. A plant of lettuce cultivar Red Crispita, arepresentative sample of seed having been deposited under ATCC AccessionNo. ______.
 3. A lettuce plant, or a part thereof, having essentiallyall of the physiological and morphological characteristics of thelettuce plant of claim
 2. 4. A progeny lettuce plant of the plant ofclaim 2 that comprises at least 50% of the alleles of the plant of claim2, wherein the progeny lettuce plant has a red mature leaf color andresistances to Bremia lactucae races 1-18 and 20-32.
 5. A seed thatproduces the plant of claim
 4. 6. A plant part of the lettuce plant ofclaim
 2. 7. The plant part of claim 6, wherein the plant part is a leaf,pollen, an ovule, an anther, a root, or a cell.
 8. A tissue culture ofregenerable cells of the plant of claim
 2. 9. A lettuce plantregenerated from the tissue culture of claim 8 or a selfed progenythereof, wherein said lettuce plant expresses essentially all of thephysiological and morphological characteristics of lettuce cultivar RedCrispita.
 10. A processed product from the plant of claim 2, wherein theprocessed product comprises cut, sliced, ground, pureed, dried, canned,jarred, washed, packaged, frozen and/or heated leaves.
 11. A method ofproducing lettuce seed, the method comprising crossing the plant ofclaim 2 with itself or a second lettuce plant and harvesting theresulting seed.
 12. A lettuce seed from a plant produced by the methodof claim
 11. 13. A lettuce plant, or part thereof, produced by growingthe seed of claim
 12. 14. A doubled haploid plant produced from thelettuce plant of claim
 13. 15. A method for producing a seed of alettuce plant derived from the plant of claim 2, the method comprising:(a) crossing a plant of lettuce cultivar Red Crispita with a secondlettuce plant; and (b) allowing seed to form; (c) growing a plant fromthe seed of step (b) to produce a plant derived from lettuce cultivarRed Crispita; (d) selfing the plant of step (c) or crossing it to asecond lettuce plant to form additional lettuce seed derived fromlettuce cultivar Red Crispita; and (e) optionally repeating steps (c)and (d) one or more times to generate further derived lettuce seed fromlettuce cultivar Red Crispita, wherein in step (c) a plant is grown fromthe additional lettuce seed of step (d) in place of growing a plant fromthe seed of step (b).
 16. A seed produced by the method of claim 15,wherein the seed comprises at least 50% of the alleles of lettucecultivar Red Crispita and is within one breeding cross of lettucecultivar Red Crispita, and wherein the seed produces a lettuce plantthat has a red mature leaf color and resistances to Bremia lactucaeraces 1-18 and 20-32.
 17. A plant, or part thereof, produced by growingthe seed of claim
 16. 18. A method of vegetatively propagating the plantof claim 2, the method comprising: (a) collecting tissue capable ofbeing propagated from a plant of lettuce cultivar Red Crispita; (b)cultivating the tissue to obtain proliferated shoots; (c) rooting theproliferated shoots to obtain rooted plantlets; and (d) optionally,growing plants from the rooted plantlets.
 19. Lettuce plantlet or plantobtained by the method of claim 18, wherein the lettuce plantlet orplant expresses essentially all of the physiological and morphologicalcharacteristics of lettuce cultivar Red Crispita.
 20. A method ofintroducing a desired added trait into lettuce cultivar Red Crispita,the method comprising: (a) crossing the plant of claim 2 with a lettuceplant that comprises a desired added trait to produce F1 progeny; (b)selecting an F1 progeny that comprises the desired added trait; (c)crossing the selected F1 progeny with lettuce cultivar Red Crispita toproduce backcross progeny; (d) selecting a backcross progeny comprisingthe desired added trait and essentially all of the physiological andmorphological characteristics of the lettuce cultivar Red Crispita; and(e) optionally repeating steps (c) and (d) one or more times to producea plant derived from lettuce cultivar Red Crispita comprising a desiredadded trait and essentially all of the physiological and morphologicalcharacteristics of lettuce cultivar Red Crispita, wherein in step (c)the selected backcross progeny produced in step (d) is used in place ofthe selected F1 progeny of step (b).
 21. The method of claim 20, whereinthe desired added trait is male sterility, pest resistance, insectresistance, disease resistance, herbicide resistance, or any combinationthereof.
 22. A lettuce plant produced by the method of claim 20 or aselfed progeny thereof, wherein the lettuce plant has the desired addedtrait.
 23. Seed of the plant of claim 22, wherein the seed produces aplant that has the desired added trait.
 24. Seed that produces the plantof claim
 22. 25. A method of producing a plant of lettuce cultivar RedCrispita comprising a desired added trait, the method comprisingintroducing a transgene conferring the desired trait into the plant ofclaim
 2. 26. A lettuce plant produced by the method of claim 25 or aselfed progeny thereof, wherein the lettuce plant has the desired addedtrait.
 27. Seed of the plant of claim 26, wherein the seed produces aplant that has the desired added trait.
 28. A method of determining agenotype of lettuce cultivar Red Crispita, the method comprising: (a)obtaining a sample of nucleic acids from the plant of claim 2; and (b)detecting a polymorphism in the nucleic acid sample.
 29. A method ofproducing a lettuce leaf, the method comprising: (a) growing the lettuceplant according to claim 2 to produce a lettuce leaf; and (b) harvestingthe lettuce leaf.
 30. A method of producing a lettuce leaf, the methodcomprising: (a) growing the lettuce plant according to claim 22 toproduce a lettuce leaf; and (b) harvesting the lettuce leaf.